Program Director/Principal Investigator (Last, First, Middle): GUO, Zhongwu Synthetic and Biological Studies of GPI Conjugates and GPI Anchorage to Cell Membranes Attaching glycosylphosphatidylinositols (GPIs), a family of complex glycolipids, to the protein C-terminus is a common and important posttranslational modification, which serves to anchor proteins to the extracellular surface. GPIs and GPI-anchored proteins/glycoproteins play a critical role in various biological and pathological processes. However, in-depth investigation of GPIs and GPI-anchored proteins is challenging because of the lack of proper methods to access and tools to study these structurally complex, diverse, and amphipathic molecules. This research project aims to address the challenge by establishing novel synthetic methods to enable access to structurally homogeneous and defined GPI-linked proteins/glycoproteins and related analogs and developing new tools and strategies to facilitate the isolation and investigation of GPI-anchored proteins/glycoproteins and GPI-binding molecules, so as to fill the gaps in GPI research. Accordingly, the future research directions of this project will include: (1) development of new methods for the synthesis of natural GPI-linked proteins based on GPI transamidase-catalyzed enzymatic GPI/protein ligation and traceless Staudinger reaction- and thio acid/azide amination-derived chemoselective GPI/protein ligations; (2) study of GPI organization and orientation on the cell surface by fluorescence resonance energy transfer (FRET) technology employing chromophore-labeled GPIs as molecular tools; (3) discovery and investigation of cell membrane components that bind/interact with GPIs using GPI derivatives that carry a photoactivatable affinity probe to pull down GPI-binding molecules; (4) qualitative and quantitative analysis of GPI-anchored proteins expressed by various cells through metabolic engineering of GPI biosynthetic pathways to facilitate the labeling and pull-down of GPI-anchored proteins for their rapid isolation, characterization, and analysis. The proposed research is innovative and will have a significant and broad impact, as it addresses a series of important but unsolved problems in GPI research and fills the gaps in understanding GPI anchorage. Specifically, a practical synthetic method for GPI-anchored proteins and glycoproteins will allow for access to these important molecules and their functionalized analogs in structurally homogeneous and defined forms, which are useful for various biological studies. Systematic and in-depth investigation of GPI-anchored proteomics, GPI organization and orientation on the cell surface, and GPI interaction with other molecules in the cell membrane, which will be enabled by the molecular probes and strategies proposed herein, will lead to a better and deeper understanding of GPI anchorage. This will not only help reveal more details about the functions and functional mechanisms of GPIs but also help discover new disease markers, including both GPI-anchored proteins/glycoproteins and GPI- binding molecules, thereby to fulfill the promise of GPI application to molecular medicine, such as development of new diagnostic and therapeutic methods.